JP2023541845A - Materials and methods for reducing protein aggregation - Google Patents

Abstract

Provided herein are methods of treating a neoplastic disease in a subject. In an exemplary embodiment, the method includes administering an aqueous solution comprising an anti-EGFRvIII agent to the subject using an administration system, and one or more of the components of the administration system, or portion thereof, that contact the aqueous solution include: Substantially free of polyvinyl chloride (PVC) and/or air. In an exemplary aspect, the administration system is an infusion system that includes an infusion line, at least a portion of which is substantially devoid of PVC. In an exemplary embodiment, the administration system includes a container for containing an aqueous solution, and when the container contains an aqueous solution, less than about 5% of the volume of the container is air. Additionally, related kits are provided herein.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS The benefit under 35 U.S.C. 119(e) of U.S. Provisional Patent Application No. 63/077,088 filed September 11, 2020 is hereby claimed; The disclosure of that provisional patent application is incorporated herein by reference.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY The computer-readable nucleotide/amino acid sequence listing filed contemporaneously with this specification is incorporated by reference in its entirety as of: September 10, 2021; It is identified as a 122,414 byte ASCII (text) file named "A-2651-WO-PCT_Sequence_Listing.txt."

The present invention relates to the administration of anti-EGFRvIII agents to treat neoplastic diseases, such as cancer.

Glioblastoma (GBM) belongs to a group of highly malignant brain tumors and represents one of the most deadly human cancers. The age-adjusted incidence of glioblastoma ranges from 0.59 to 3.69 per 100,000 people worldwide. Despite aggressive surgical, radiological, and chemotherapeutic interventions, tumors progress within months or weeks, and overall survival has decreased since FDA approval of temozolomide (TMZ) in 2005. The period is 12 to 15 months, and there is almost no change in prognosis (Omuro & DeAngelis, JAMA, 2013; 310:1842-1850).

If recurrence occurs after primary surgery, treatment of glioblastoma depends on age, clinical status, histology, response to initial treatment, time since initial diagnosis, and whether the occurrence is localized or diffuse. . Palliative care is a common choice for patients with diffuse or multifocal tumor recurrence. For patients with localized disease, a combination of surgery, nitrosourea-based therapy, and radiation (standard re-irradiation or highly conformal radiation) is used, with poor results. After failure to respond to two consecutive drugs, the likelihood of responding to chemotherapy decreases (Stewart et al., Lancet 2002;359(9311):1011-1018). Furthermore, no new drug has demonstrated a survival benefit from randomized clinical trials (Mehta et al, Crit Rev Oncol Hematol. 2017; 111:60-65).

EGFR expression and enhanced EGF pathway signaling activity following amplification of the gene encoding the epidermal growth factor receptor (EGFR) have been shown to be associated with glioblastomas (almost exclusively isocitrate dehydrogenase (IDH) wild-type glioblastomas). (Louis et al., Acta Neuropathol. 2016; 131:803-820). Approximately 50% of glioblastomas are EGFR amplification positive, and half of these have a concomitant glioblastoma that encodes a truncated, constitutively active receptor called EGFRvIII (epidermal growth factor receptor variant III). Expresses the EGFR mutation. Similar to native EGFR, the mutant EGFRvIII is a membrane-bound receptor; however, the deletion includes an extracellular ligand-binding domain and is characterized by a new glycine residue occurring at the splice junction. This results in a protein lacking 267 amino acid residues (Wong et al., Proc Natl Acad Sci USA. 1992; 89:2965-2969). Although lacking an extracellular ligand-binding domain, EGFRvIII exhibits a ligand-independent constitutive tyrosine kinase activity that promotes malignant growth by stimulating downstream signaling pathways (Mellinghoff et al., N. Engl J Med. 2005; 353: 2012-2024). According to a meta-analysis (Chen et al., Acta Neurol Scand. 2015; 132:310-322), there is currently no evidence that either EGFR amplification or EGFRvIII mutations have prognostic value in glioblastoma patients. However, it is insufficient. Nevertheless, EGFRvIII is considered to be a bona fide tumor-specific antigen found exclusively on tumor cells, making it an interesting anti-tumor therapeutic strategy.

Omuro & DeAngelis, JAMA, 2013; 310: 1842-1850 Stewart et al. , Lancet 2002;359(9311):1011-1018 Mehta et al, Crit Rev Oncol Hematol. 2017;111:60-65 Louis et al. , Acta Neuropathol. 2016;131:803-820 Wong et al. , Proc Natl Acad Sci USA. 1992;89:2965-2969 Mellinghoff et al. , N Engl J Med. 2005;353:2012-2024 Chen et al. , Acta Neurol Scand. 2015;132:310-322

Those skilled in the art, based on the disclosure provided herein, will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Will. Such equivalents are intended to be encompassed by embodiment (E) below.
E1. A method of treating a neoplastic disease in a subject, the method comprising administering to the subject an aqueous solution containing an anti-EGFRvIII agent using an administration system, the method comprising: administering to the subject an aqueous solution containing an anti-EGFRvIII drug, the administration system comprising: A method in which one or more portions of the element are substantially devoid of polyvinyl chloride (PVC) or PVC plasticizer and/or substantially devoid of air.
E2. The method according to E1, wherein the administration system is an infusion system.
E3. The method of E2, wherein the injection system includes an injection line, and at least a portion of the injection line is substantially free of PVC or PVC plasticizer.
E4. The method of E3, wherein the portion of the injection line in contact with the aqueous solution is substantially devoid of PVC, and optionally the inner surface of the injection line in contact with the aqueous solution is substantially devoid of PVC or PVC plasticizer.
E5. The method of E3 or E4, wherein the injection line comprises polyolefin elastomer (POE) or polyurethane (PUR) or ethylene vinyl acetate (EVA).
E6. The method according to E5, wherein the injection line consists entirely of POE, PUR, or EVA, or the inner surface of the injection line is coated with POE, PUR, or EVA.
E7. A method as in any one of the preceding embodiments, wherein the administration system includes a container for containing an aqueous solution.
E8. The method according to E7, wherein at least a portion of the container is substantially devoid of PVC or PVC plasticizer, and optionally a portion of the container that contacts the aqueous solution is substantially devoid of PVC or PVC plasticizer.
E9. A method according to E7 or E8, wherein when the container contains an aqueous solution, the container is substantially devoid of air.
E10. The method of E8, wherein when the container contains an aqueous solution, less than about 5% of the volume of the container is air.
E11. The method of E9, wherein when the container contains an aqueous solution, less than about 3% of the volume of the container is air.
E12. A method according to any one of the preceding embodiments, wherein the aqueous solution is administered to the subject by intravenous (IV) infusion, optionally continuous intravenous infusion (cIVi).
E13. The method of E12, wherein the anti-EGFRvIII drug is administered to the subject as a constant flow cIVi.
E14. The method of any one of E2-E13, wherein the infusion system further comprises an infusion line filter, an intravenous (IV) bag, an infusion pump, or a combination thereof.
E15. The method of E14, wherein the infusion line filter is a polyethylsulfone (PES) filter.
E16. The method of E14 or E15, wherein when the IV bag comprises an aqueous solution, the IV bag is substantially devoid of air.
E17. The method of E16, wherein less than about 5% of the volume of the IV bag is air when the container contains an aqueous solution.
E18. The method of E17, wherein less than about 3% of the volume of the IV bag is air when the container contains an aqueous solution.
E19. Any one of E14-E18, wherein at least a portion of the IV bag is substantially devoid of PVC or PVC plasticizer, and optionally, a portion of the IV bag that contacts the aqueous solution is substantially devoid of PVC or PVC plasticizer. The method described in.
E20. The method of E19, wherein at least a portion of the IV bag comprises ethylene vinyl acetate (EVA) or polyolefin elastomer (POE).
E21. The method according to E20, wherein the IV bag consists entirely of EVA or POE, or the inner surface of the IV bag is lined with EVA or POE.
E22. The method according to any one of E14-E21, wherein the infusion pump is a CADD pump.
E23. The method of any one of E14-E22, wherein the infusion pump is programmable, lockable, non-elastomeric, and/or has an alarm.
E24. A method according to any one of the preceding embodiments, wherein the aqueous solution is injected at an injection rate of about 1.5 mL/hour to about 150 mL/hour, optionally about 1.5 mL/hour to about 5 mL/hour.
E25. The method of any one of the preceding embodiments, wherein the average cumulative count of subvisible particles per mL of solution after administration is less than 15 as measured by a submerged particle counter.
E26. The method of E25, wherein the average cumulative count of subvisible particles per mL of solution after administration is less than 10, optionally less than 1, as measured by a submerged particle counter.
E27. A method as in any one of the preceding embodiments, wherein upon visual inspection, the solution after administration has less than 5 visible particles of 125 μm or larger.
E28. The method according to any one of the preceding embodiments, wherein the proportion of high molecular weight (HMW) species in the solution after administration is less than 5% as determined by size exclusion high performance liquid chromatography (SE-HPLC). .
E29. The proportion of high molecular weight (HMW) species in the solution after administration is less than 2% and/or the proportion of the main peak of the anti-EGFRvIII drug is greater than 95%, as determined by SE-HPLC. A method as in any one of the embodiments.
E30. The practice as described above, wherein the recovery of the anti-EGFRvIII drug in solution after administration is greater than 95% as determined by ultraviolet-visible (UV-VIS) spectroscopy or reversed-phase high performance liquid chromatography (RP-HPLC). A method according to any one of the embodiments.
E31. A method according to any one of the preceding embodiments, wherein the neoplastic disease is cancer or a tumor.
E32. The method according to E31, wherein the cancer or tumor is an EGFRvIII-positive cancer or tumor.
E32. The method according to E31 or E32, wherein the cancer is a squamous cell tumor such as non-small cell lung cancer (NSCLC).
E33. The method according to E31 or E32, wherein the cancer is glioblastoma or malignant glioma.
E34. The method of any one of the embodiments, wherein the aqueous solution comprises saline, optionally about 0.5% to about 1.5% (v/v) saline.
E35. The aforementioned embodiments, wherein the solution comprises about 2% (v/v) to about 6% (v/v) intravenous saline solution (IVSS), optionally about 4% (v/v) IVSS. The method described in any one of .
E36. A method according to any one of the preceding embodiments, wherein the anti-EGFRvIII drug is present in the aqueous solution at a concentration of less than 75 ng/mL, optionally less than 50 ng/mL.
E37. The method of E36, wherein the anti-EGFRvIII drug is present in the aqueous solution at a concentration of about 75 ng/mL to about 500 ng/mL, optionally about 200 ng/mL to about 400 ng/mL.
E38. A method as in any one of the preceding embodiments, wherein the final volume of the aqueous solution is about 100 mL to about 300 mL of solution.
E39. The aforementioned embodiments, wherein the anti-EGFRvIII drug is a bispecific antigen binding polypeptide and comprises a first binding domain that binds human EGFRvIII and macaque EGFRvIII, and a second binding domain that binds human CD3. The method described in any one of .
E40. The method of E39, wherein the human EGFRvIII comprises the amino acid sequence of SEQ ID NO: 1 and the macaque EGFRvIII comprises the amino acid sequence of SEQ ID NO: 2.
E41. The method according to E39 or E40, wherein said human CD3 comprises residues 1-27 of SEQ ID NO: 123.
E42. The method according to any one of E39-E41, wherein said human CD3 comprises the amino acid sequence of SEQ ID NO: 123.
E43. The EGFRvIII binding domain comprises (a) (i) VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 3; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 4; and (iii) ) A heavy chain variable region (VH) comprising CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5; (b) (i) VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 6; ( Any one of E39-42, comprising: ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7; and (iii) a light chain variable region (VL) comprising CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8. The method described in.
E44. The method according to any one of E39-E43, wherein the EGFRvIII binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 9 and a VL comprising the amino acid sequence of SEQ ID NO: 10.
E45. The method according to E43 or E44, wherein the VH and VL are connected by a linker to form a single chain Fv (scFv).
E46. The method according to E45, wherein the linker is a peptide linker comprising a sequence selected from any one of SEQ ID NOs: 114-122.
E47. The method of E45 or E46, wherein the linker comprises (Gly4Ser)x, where x is an integer of 1, 2, 3, or 4.
E48. The method according to any one of E39-E47, wherein the EGFRvIII binding domain comprises the amino acid sequence of SEQ ID NO: 11.
E49. The CD3 binding domain is
a. VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 18, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 19, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 20; CDR comprising the amino acid sequence of SEQ ID NO: 15; - a VL comprising L1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;
b. A VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 27, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 28, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 29; CDR comprising the amino acid sequence of SEQ ID NO: 24. - a VL comprising L1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 25, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 26;
c. A VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 36, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 37, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 38; and a CDR comprising the amino acid sequence of SEQ ID NO: 33. - a VL comprising L1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 34, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35;
d. A VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 45, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 46, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 47; CDR comprising the amino acid sequence of SEQ ID NO: 42. - a VL comprising L1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 43, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 44;
e. A VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 54, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 55, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 56; CDR comprising the amino acid sequence of SEQ ID NO: 51. - a VL comprising L1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 52, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 53;
f. A VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 63, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 64, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 65; CDR comprising the amino acid sequence of SEQ ID NO: 60. - a VL comprising L1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 61, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 62;
g. VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 72, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 73, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 74; CDR comprising the amino acid sequence of SEQ ID NO: 69 - a VL comprising L1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 70, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 71;
h. A VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 81, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 82, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 83; and a CDR comprising the amino acid sequence of SEQ ID NO: 78. - a VL comprising L1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 79, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 80;
i. VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 90, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 91, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 92; CDR comprising the amino acid sequence of SEQ ID NO: 87 - a VL comprising L1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 88, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 89;
j. VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 99, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 100, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 101; CDR comprising the amino acid sequence of SEQ ID NO: 96 -L1, a VL comprising CDR-L2 comprising the amino acid sequence of SEQ ID NO: 97, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 98; or k. A VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 108, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 109, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 110; CDR comprising the amino acid sequence of SEQ ID NO: 105. -L1, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 106, and a VL comprising CDR-L3 comprising the amino acid sequence of SEQ ID NO: 107.
E50. The CD3 binding domain is
a. A VH comprising the amino acid sequence of SEQ ID NO: 21, and a VL comprising the amino acid sequence of SEQ ID NO: 22;
b. A VH comprising the amino acid sequence of SEQ ID NO: 30 and a VL comprising the amino acid sequence of SEQ ID NO: 31;
c. VH comprising the amino acid sequence of SEQ ID NO: 39, and VL comprising the amino acid sequence of SEQ ID NO: 40;
d. A VH comprising the amino acid sequence of SEQ ID NO: 48 and a VL comprising the amino acid sequence of SEQ ID NO: 49;
e. VH comprising the amino acid sequence of SEQ ID NO: 57, and VL comprising the amino acid sequence of SEQ ID NO: 58;
f. VH comprising the amino acid sequence of SEQ ID NO: 66, and VL comprising the amino acid sequence of SEQ ID NO: 67;
g. VH comprising the amino acid sequence of SEQ ID NO: 75, and VL comprising the amino acid sequence of SEQ ID NO: 76;
h. VH comprising the amino acid sequence of SEQ ID NO: 84, and VL comprising the amino acid sequence of SEQ ID NO: 85;
i. VH comprising the amino acid sequence of SEQ ID NO: 93, and VL comprising the amino acid sequence of SEQ ID NO: 94;
j. a VH comprising the amino acid sequence of SEQ ID NO: 102; a VL comprising the amino acid sequence of SEQ ID NO: 103; or k. The method according to any one of E39 to E49, comprising a VH comprising the amino acid sequence of SEQ ID NO: 111 and a VL comprising the amino acid sequence of SEQ ID NO: 112.
E51. The method according to any one of E49 or E50, wherein the VH and VL of the CD3 binding domain are connected by a linker to form a single chain Fv (scFv).
E52. The method according to E51, wherein the linker is a peptide linker comprising a sequence selected from any one of SEQ ID NOs: 114-122.
E53. The method of E51 or E52, wherein the linker comprises (Gly4Ser)x, where x is an integer of 1, 2, 3, or 4.
E54. Any one of E39 to E53, wherein the CD3 binding domain comprises the amino acid sequence of any one of SEQ ID NOs: 23, 32, 41, 50, 59, 68, 77, 86, 95, 104, and 113. the method of.
E55.
The EGFRvIII binding domain comprises (a) (i) VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 3; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 4; and (iii) ) A heavy chain variable region (VH) comprising CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5; (b) (i) VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 6; ( ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7; and (iii) a light chain variable region (VL) comprising CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8;
The CD3 binding domain comprises (a) (i) VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 99; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 100; and (iii) ) A heavy chain variable region (VH) comprising CDR-H3 comprising the amino acid sequence of SEQ ID NO: 101; (b) (i) VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 96; ( Any one of E39 to E54, comprising: ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 97; and (iii) a light chain variable region (VL) comprising CDR-L3 comprising the amino acid sequence of SEQ ID NO: 98. The method described in.
E56. The EGFRvIII binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 9 and a VL comprising the amino acid sequence of SEQ ID NO: 10, and the CD3 binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 102, and a VH comprising the amino acid sequence of SEQ ID NO: 103. and a VL comprising the amino acid sequence of E39-E55.
E57. The method of any one of E39-E56, wherein the EGFRvIII binding domain comprises the amino acid sequence of SEQ ID NO: 11 and the CD3 binding domain comprises the amino acid sequence of SEQ ID NO: 104.
E58. The method according to any one of E39-E57, wherein the EGFRvIII binding domain and the CD3 binding domain are connected by a linker.
E59. The method according to E58, wherein the linker is a peptide linker comprising a sequence selected from any one of SEQ ID NOs: 114-122.
E60. The method of E58 or E59, wherein the linker comprises (Gly4Ser)x, where x is an integer of 1, 2, 3, or 4.
E61. 12. The method of any one of the preceding embodiments, wherein the anti-EGFRvIII drug comprises the amino acid sequence of SEQ ID NO: 12.
E62. 13. The method of any one of the preceding embodiments, wherein the anti-EGFRvIII drug comprises the amino acid sequence of SEQ ID NO: 13.
E63. The method of any one of E1-E62, wherein the anti-EGFRvIII drug is administered at an initial dose of about 15 μg/day to about 12000 μg/day for at least 14 days.
E64. The method of any one of E1-E63, wherein the anti-EGFRvIII drug is administered at an initial dose of about 1500 μg/day to about 6000 μg/day for at least 14 days.
E65. The method of any one of E1-E64, wherein the anti-EGFRvIII drug is administered at an initial dose of about 3000 μg/day to about 6000 μg/day for at least 14 days.
E66. The method of any one of E1-E65, wherein the anti-EGFRvIII drug is administered at an initial dose of about 15 μg/day to about 12000 μg/day for at least 28 days.
E67. The method of any one of E1-E66, wherein the anti-EGFRvIII drug is administered at an initial dose of about 1500 μg/day to about 6000 μg/day for at least 28 days.
E68. The method of any one of E1-E67, wherein the anti-EGFRvIII drug is administered at an initial dose of about 3000 μg/day to about 6000 μg/day for at least 28 days.
E69. The method of any one of E1-E68, wherein the anti-EGFRvIII drug is administered in a 14 day on/14 day off cycle or a 28 day on/14 day off cycle.
E70. A kit comprising a delivery system and an anti-EGFRvIII agent, wherein one or more of the components, or at least a portion of the components, of the delivery system that contacts the aqueous solution substantially contains PVC or PVC plasticizer and/or air. Missing kit.
E71. The kit of E70, wherein the administration system is an infusion system comprising an infusion line, at least a portion of which is substantially free of PVC or PVC plasticizer.
E72. The kit according to E70 or E71, wherein the administration system includes a container for containing an aqueous solution, and when the container contains an aqueous solution, less than about 5% of the volume of the container is air.
E73. An article of manufacture comprising an aqueous solution containing an anti-EGFRvIII drug within a container, wherein less than about 5% of the volume of the container is air when the container contains the aqueous solution.
E74. A kit comprising an IV bag containing an aqueous solution comprising about 3% (v/v) to about 6% (v/v) IVSS and saline, and a container containing an anti-EGFRvIII drug, the kit being in contact with the aqueous solution. A kit wherein at least a portion of the IV bag is substantially devoid of PVC or PVC plasticizer.

FIG. 3 is an image of particulation of anti-EGFRvIII drug (AMG 596) in an IV bag after stirring. Images of an aqueous solution containing anti-EGFRvIII drug (AMG 596) in an IV bag (250 mL MediBag) without air (Figure 2A) or with air (Figure 2B). Images of an aqueous solution containing anti-EGFRvIII drug (AMG 596) in an IV bag (250 mL MediBag) without air (Figure 2A) or with air (Figure 2B). FIG. 3 is a graph of particle counts per mL of an aqueous solution containing AMG 596 in the indicated IV bags versus the indicated particle sizes, respectively. FIG. FIG. 3 is a graph of particle counts per mL of an aqueous solution containing AMG 596 in the indicated IV bags versus the indicated particle sizes, respectively. FIG. Figure 2 is a graph of protein recovery of AMG 596 in indicated IV bags with and without air. Recovery of AMG 596 was nearly 100% for all IV bags when no air was present in the IV bags. 1 is a flowchart of the experimental steps according to an aqueous solution containing an anti-EGFRvIII drug (AMG 596) as described herein. Figure 2 is a table listing details of IV bag preparation. Figure 3 is a graph of cumulative particle counts per mL of an aqueous solution containing an anti-EGFRvIII drug (AMG 596) passed through an infusion system with and without an in-line filter, respectively. The particle counts for 10 μm particles are shown in FIG. 4A, and the particle counts for 25 μm particles are shown in FIG. 4B. Figure 3 is a graph of cumulative particle counts per mL of an aqueous solution containing an anti-EGFRvIII drug (AMG 596) passed through an infusion system with and without an in-line filter, respectively. The particle counts for 10 μm particles are shown in FIG. 4A, and the particle counts for 25 μm particles are shown in FIG. 4B. Figure 2 is a graph of cumulative particle counts per mL of an aqueous solution containing an anti-EGFRvIII drug (AMG 596) passed through an infusion system at an infusion rate of 5 mL/hour or 125 mL/hour, respectively. The particle counts for 25 μm particles are shown in FIG. 5A, and the particle counts for 10 μm particles are shown in FIG. 5B. Figure 2 is a graph of cumulative particle counts per mL of an aqueous solution containing an anti-EGFRvIII drug (AMG 596) passed through an infusion system at an infusion rate of 5 mL/hour or 125 mL/hour, respectively. The particle counts for 25 μm particles are shown in FIG. 5A, and the particle counts for 10 μm particles are shown in FIG. 5B.

1. Overview AMG 596 is a bispecific T cell inducing protein that activates T cells by binding to EGFRvIII and CD3, allowing them to access and kill glioblastoma cells. AMG 596 is a very potent molecule with a short half-life. Because this protein has a very short half-life, it is administered by continuous intravenous infusion (cIVi) to maintain sufficient levels of protein to be effective in the patient. A cIVi administration setup typically consists of at least an IV bag, an infusion pump, and an infusion line. In some embodiments, the AMG 596 formulation (DP) is mixed in an IV bag with saline and an intravenous solution stabilizer (IVSS) to form an infusion solution. This solution is then infused into the patient by pumping it into the patient through an infusion line with an infusion pump. The present disclosure provides an efficient and improved method of administering AMG 596 to treat patients with neoplastic diseases based on the evaluation of infusion systems and their components and materials.

2. Definitions An antigen-binding polypeptide according to the invention is preferably a polypeptide that immunospecifically binds to its target, ie the antigen. The polypeptide typically includes domains that include or are derived from the heavy chain variable region (VH) and/or light chain variable region (VL) of an antibody. Polypeptides according to the invention contain the minimal structural requirements of antibodies that allow immunospecific target binding. This minimum requirement may include, for example, at least three light chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VL region) and/or three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VH region), preferably six. It can be defined by the presence of all CDRs. Where (and in what order) the CDRs are located within the binding domain is within the knowledge of those skilled in the art.

T cell-inducing antigen-binding polypeptides according to the invention, such as BiTE® molecules, are preferably bispecific, as defined herein typically with one domain that binds at least one target antigen; , and another domain that binds to CD3. Therefore, this antigen-binding polypeptide is not naturally occurring and its function is significantly different from natural products. A polypeptide according to the invention is therefore an artificial "hybrid" polypeptide comprising at least two separate binding domains with different specificities and is therefore bispecific. The bispecific T cell-inducing polypeptides disclosed herein may contain three or more domains, e.g., two identical or different target antigen binding domains and another domain that binds CD3. It is emphasized that it may include The target antigen binding domains may be identical, ie, bind to the same epitope, or they may bind to different epitopes of the same or different target antigens. A T cell inducing polypeptide may be characterized by the presence of 3 to 6 CDRs in either or both binding domains.

Exemplary bispecific anti-EGFRvIII agents (e.g., BiTE® molecules) disclosed herein are antigen-binding polypeptides that include two or more antigen-binding domains, with one domain binding to EGFRvIII. and another domain binds to CD3. In an exemplary embodiment, each binding domain comprises an scFv (the VL and VH domains of a single arm of an antibody joined via a linker). Although germline VL and VH domains are encoded by separate genes, they can be joined using recombinant methods by synthetic linkers that can create them as a single protein chain; In this case, the VL and VH regions are paired to form a monovalent molecule (known as a single chain Fv (scFV)); see, for example, Bird et al. Science 242:423-426 (1988) and Huston et al. , 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883.

"Variable domain" refers to the variable region of an antibody light chain (VL) or the variable region of an antibody heavy chain (VH), either alone or in combination. As is known in the art, the heavy and light chain variable regions each consist of four framework regions (FRs) linked by three complementarity determining regions (CDRs) and are Contributes to the formation of antigen binding sites.

Exemplary EGFRvIII binding domain and CD3 binding domain "complementarity determining regions" (CDRs) are set forth in the sequence listing. CDRs may be defined according to Kabat, Chothia, both Kabat and Chothia accumulation, AbM, contact, North, and/or conformational definitions, or any method for determining CDRs well known in the art. can. For example, Kabat et al. , 1991, Sequences of Proteins of Immunological Interest, 5th ed. (hypervariable region); Chothia et al. , 1989, Nature 342:877-883 (structural loop structures). The AbM definition of CDRs is a compromise between Kabat and Chothia and uses Oxford Molecular's AbM antibody modeling software (Accelrys®). The identity of the amino acid residues of a particular antibody that make up the CDRs can be determined using methods well known in the art.

"About" or "approximately", when used in reference to a measurable numerical variable, refers to the indicated value of this variable and within experimental error of this indicated value (e.g., within the 95% confidence interval of the mean) or ±10% of this indicated value, whichever is greater. Numeric ranges include the numbers that define the range.

3. Anti-EGFRvIII Drugs The epidermal growth factor receptor (EGFR) is an important regulator of normal cell growth in tissues of epithelial origin. Dysregulation of EGFR signaling, resulting from mechanisms such as cell surface overexpression, autocrine activation, and EGFR gene mutations, contributes to the formation of many epithelial malignancies in humans. Several EGFR mutations have been described. The most common extracellular mutation is EGFRvIII (also known as de2-7EGFR and ΔEGFR). EGFRvIII is a tumor-specific mutation resulting from an 801 base pair in-frame deletion spanning exons 2-7 of the coding sequence. This deletion removes 267 amino acids from the extracellular domain, creating a binding site between exons 1 and 8 and a new glycine residue. EGFRvIII has a molecular weight of approximately 145 kDa. The amino acid sequences of human and cynomolgus monkey EGFRvIII are shown as SEQ ID NO: 1 and 2, respectively.

An exemplary anti-EGFRvIII drug is a bispecific molecule that binds EGFRvIII and CD3, such as a BiTE® (bispecific T cell induction) molecule. BiTE® molecules are recombinant proteins formed from two flexibly linked binding domains, each domain derived from an antibody. One binding domain of the BiTE® molecule is specific for a tumor-associated surface antigen (e.g. EGFRvIII) and the second binding domain is a subunit of the T cell receptor complex on T cells. Specific to CD3. These designs make BiTE® molecules uniquely suited to temporarily bind T cells and target cells while simultaneously potently activating the cytotoxic capacity of T cells against their natural target cells. It has become something like this. For example, see WO 99/54440 pamphlet, WO 2005/040220 pamphlet, and WO 2008/119567 pamphlet.

Thus, in some embodiments, the described anti-EGFRvIII agents have two binding domains: a first domain that binds EGFRvIII (preferably human EGFRvIII) and a second domain that binds CD3 (preferably human CD3). domain. Exemplary CD3 sequences are shown in SEQ ID NOs: 123-126. Preferably, the second domain binds residues 1-27 of SEQ ID NO: 123. Alternatively, the second domain may bind residues 1-27 of any one of SEQ ID NOs: 124-126.

In certain embodiments, the EGFRvIII binding domain comprises (a) (i) a VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 3; (ii) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 4; and (iii) a heavy chain variable region (VH) comprising CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5; and (b) (i) a VL complementarity determining region 1 (CDR) comprising the amino acid sequence of SEQ ID NO: 6. -L1); (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7; and (iii) a light chain variable region (VL) comprising CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8.

In certain embodiments, the EGFRvIII binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 9 and a VL comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the VH and VL are joined by a linker to form a single chain Fv (scFv). In some embodiments, the linker is a peptide linker comprising a sequence selected from any one of SEQ ID NOs: 114-122. In some embodiments, the linker is a GS linker, such as Gly-Gly-Gly-Gly-Ser (G4S, SEQ ID NO: 115) or a polymer thereof, i.e. (Gly4Ser)x, where x is one or more (eg, 2 or 3) (eg, SEQ ID NO: 121, 122).

In certain embodiments, the EGFRvIII binding domain comprises the amino acid sequence of SEQ ID NO:11.

In certain embodiments, the CD3 binding domain is
(a) VH containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 18, CDR-H2 containing the amino acid sequence of SEQ ID NO: 19, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 20; and the amino acid sequence of SEQ ID NO: 15. A VL comprising CDR-L1 comprising CDR-L1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 16, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 17;
(b) VH containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 27, CDR-H2 containing the amino acid sequence of SEQ ID NO: 28, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 29; and the amino acid sequence of SEQ ID NO: 24. A VL comprising CDR-L1 comprising CDR-L2 comprising the amino acid sequence of SEQ ID NO: 25, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 26;
(c) VH containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 36, CDR-H2 containing the amino acid sequence of SEQ ID NO: 37, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 38; and the amino acid sequence of SEQ ID NO: 33. A VL comprising CDR-L1 comprising CDR-L1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 34, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 35;
(d) VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 45, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 46, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 47; and the amino acid sequence of SEQ ID NO: 42. A VL comprising CDR-L1 comprising CDR-L1 comprising, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 43, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 44;
(e) VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 54, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 55, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 56; and the amino acid sequence of SEQ ID NO: 51. a VL comprising CDR-L1 comprising CDR-L2 comprising the amino acid sequence of SEQ ID NO: 52, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 53;
(f) VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 63, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 64, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 65; and the amino acid sequence of SEQ ID NO: 60. A VL comprising CDR-L1 comprising CDR-L1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 61, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 62;
(g) VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 72, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 73, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 74; and the amino acid sequence of SEQ ID NO: 69. A VL comprising CDR-L1 comprising CDR-L1 comprising, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 70, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 71;
(h) VH containing CDR-H1 containing the amino acid sequence of SEQ ID NO: 81, CDR-H2 containing the amino acid sequence of SEQ ID NO: 82, and CDR-H3 containing the amino acid sequence of SEQ ID NO: 83; and the amino acid sequence of SEQ ID NO: 78. A VL comprising CDR-L1 comprising CDR-L2 comprising the amino acid sequence of SEQ ID NO: 79, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 80;
(i) VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 90, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 91, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 92; and the amino acid sequence of SEQ ID NO: 87. A VL comprising CDR-L1 comprising CDR-L1, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 88, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 89;
(j) VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 99, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 100, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 101; and the amino acid sequence of SEQ ID NO: 96. or (k) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 108; , a VH comprising CDR-H2 comprising the amino acid sequence of SEQ ID NO: 109, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 110; CDR-L1 comprising the amino acid sequence of SEQ ID NO: 105, comprising the amino acid sequence of SEQ ID NO: 106. CDR-L2, and a VL comprising CDR-L3 comprising the amino acid sequence of SEQ ID NO: 107.

In certain embodiments, the CD3 binding domain is
(a) VH containing the amino acid sequence of SEQ ID NO: 21 and VL containing the amino acid sequence of SEQ ID NO: 22;
(b) VH comprising the amino acid sequence of SEQ ID NO: 30 and VL comprising the amino acid sequence of SEQ ID NO: 31;
(c) a VH comprising the amino acid sequence of SEQ ID NO: 39 and a VL comprising the amino acid sequence of SEQ ID NO: 40;
(d) a VH comprising the amino acid sequence of SEQ ID NO: 48 and a VL comprising the amino acid sequence of SEQ ID NO: 49;
(e) a VH comprising the amino acid sequence of SEQ ID NO: 57 and a VL comprising the amino acid sequence of SEQ ID NO: 58;
(f) a VH comprising the amino acid sequence of SEQ ID NO: 66 and a VL comprising the amino acid sequence of SEQ ID NO: 67;
(g) a VH comprising the amino acid sequence of SEQ ID NO: 75 and a VL comprising the amino acid sequence of SEQ ID NO: 76;
(h) a VH comprising the amino acid sequence of SEQ ID NO: 84 and a VL comprising the amino acid sequence of SEQ ID NO: 85;
(i) a VH comprising the amino acid sequence of SEQ ID NO: 93 and a VL comprising the amino acid sequence of SEQ ID NO: 94;
(j) a VH containing the amino acid sequence of SEQ ID NO: 102 and a VL containing the amino acid sequence of SEQ ID NO: 103; or (k) a VH containing the amino acid sequence of SEQ ID NO: 111 and a VL containing the amino acid sequence of SEQ ID NO: 112; including.

In some embodiments, the VH and VL of the CD3 binding domain are joined by a linker to form a single chain Fv (scFv). In some embodiments, the linker is a peptide linker comprising a sequence selected from any one of SEQ ID NOs: 114-122. In some embodiments, the linker is a GS linker, such as Gly-Gly-Gly-Gly-Ser (G4S, SEQ ID NO: 115) or a polymer thereof, i.e. (Gly4Ser)x, where x is one or more (eg, 2 or 3) (eg, SEQ ID NO: 121, 122).

In certain embodiments, the CD3 binding domain comprises the amino acid sequence of any one of SEQ ID NOs: 23, 32, 41, 50, 59, 68, 77, 86, 95, 104, and 113.

In certain embodiments, the EGFRvIII binding domain and the CD3 binding domain are connected by a linker. In some embodiments, the linker is a peptide linker comprising a sequence selected from any one of SEQ ID NOs: 114-122. In some embodiments, the linker is a GS linker, such as Gly-Gly-Gly-Gly-Ser (G4S, SEQ ID NO: 115) or a polymer thereof, i.e. (Gly4Ser)x, where x is one or more (eg, 2 or 3) (eg, SEQ ID NO: 121, 122).

In certain embodiments, the anti-EGFRvIII agents described herein include two domains. The first domain binds to EGFRvIII (preferably human EGFRvIII) and comprises (a) (i) a VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 3; (ii) a VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 4; CDR-H2 comprising the amino acid sequence; and (iii) a heavy chain variable region (VH) comprising CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5; (b) (i) a VL complement comprising the amino acid sequence of SEQ ID NO: 6; Sex-determining region 1 (CDR-L1); (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7; and (iii) light chain variable region (VL) comprising CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8. including. The second domain binds to CD3 (preferably human CD3) and comprises (a) (i) a VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 99; (ii) an amino acid sequence of SEQ ID NO: 100; CDR-H2 comprising the amino acid sequence; and (iii) a heavy chain variable region (VH) comprising CDR-H3 comprising the amino acid sequence of SEQ ID NO: 101; (b) (i) a VL complement comprising the amino acid sequence of SEQ ID NO: 96; Sex-determining region 1 (CDR-L1); (ii) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 97; and (iii) light chain variable region (VL) comprising CDR-L3 comprising the amino acid sequence of SEQ ID NO: 98. including.

In certain embodiments, the anti-EGFRvIII agents described herein have two domains: (a) a VH that binds EGFRvIII (preferably human EGFRvIII) and comprises the amino acid sequence of SEQ ID NO: 9; (b) a VH that binds to CD3 (preferably human CD3) and includes the amino acid sequence of SEQ ID NO: 102; and a VL that includes the amino acid sequence of SEQ ID NO: 103; and a second domain.

In certain embodiments, the anti-EGFRvIII agents described herein have two domains: (a) a first domain that binds to EGFRvIII (preferably human EGFRvIII) and comprises the amino acid sequence of SEQ ID NO: 11; (b) a second domain that binds to CD3 (preferably human CD3) and comprises the amino acid sequence of SEQ ID NO: 104.

In certain embodiments, an anti-EGFRvIII agent described herein comprises the amino acid sequence of SEQ ID NO: 12. In certain embodiments, an anti-EGFRvIII agent described herein comprises the amino acid sequence of SEQ ID NO:13.

Preferably, the anti-EGFRvIII drug is administered parenterally (eg, intravenously) and is then capable of crossing the blood-brain barrier (BBB). Without wishing to be bound by any particular theory, it is believed that binding to CD3 contributes to the BBB penetration of the exemplary anti-EGFRvIII drugs described herein. . Activated T lymphocytes are known to have the ability to cross the BBB under normal physiological conditions. By binding to CD3 on the surface of T cells, exemplary anti-EGFRvIII drugs are believed to activate peripheral circulating T cells, thereby allowing them to cross the BBB.

Methods of producing anti-EGFRvIII drugs are well known to those skilled in the art. For example, in "single chain Fv" (scFv), the two domains of the Fv fragment, VL and VH, are encoded by separate genes, but they can be made as a single protein chain ( They can be joined using recombinant methods by artificial linkers (as described above), where it is known to pair the VL and VH regions to form a monovalent molecule; e.g. , Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883. These antibody fragments and derivatives are obtained using conventional techniques known to those skilled in the art, and the fragments are evaluated for function in the same manner as full-length antibodies or IgGs. Therefore, a single chain variable fragment (scFv) is a fusion protein of an immunoglobulin heavy chain variable region (VH) and light chain variable region (VL), usually connected by a short linker peptide. Linkers are typically rich in glycine for flexibility (as discussed above), as well as serine or threonine for solubility. This protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of a linker.

Techniques described for the production of polypeptides derived from single chain antibodies (inter alia, US Pat. No. 4,946,778, Kontermann and Duebel (2010), loc.cit. and Little (2009), loc. cit.) can be adapted to generate antigen-binding polypeptides that specifically recognize the target of choice.

(scFv) _Bivalent (also called divalent) or bispecific single chain variable fragments (bi-scFv or di-scFv) that have two configurations (e.g. can be modified by linking (by a linker as described above). This linkage can be performed by creating a single chain polypeptide comprising two VH regions and two VL regions, generating a tandem scFv (e.g., Kufer P. et al., (2004) Trends in Biotechnology 22(5):238-244).

4. Methods of Treatment The present disclosure provides methods of administering anti-EGFRvIII agents to treat neoplastic disease in a subject, and methods of treating neoplastic disease in a subject. In an exemplary embodiment, the method includes administering a composition (e.g., an aqueous solution) comprising an anti-EGFRvIII agent to a subject using an administration system, the administration system contacting the composition (e.g., an aqueous solution). One or more of the components, or at least a portion of the components, are substantially devoid of polyvinyl chloride (PVC) or PVC plasticizer and/or substantially devoid of air. In an exemplary embodiment, the method includes administering a composition (e.g., an aqueous solution) comprising an anti-EGFRvIII drug to a subject using an infusion system that includes an infusion line, the infusion line being a PVC infusion line. , or substantially free of PVC or PVC plasticizer. Optionally, the injection line is constructed entirely of a material other than PVC. Alternatively, the inside of the injection line is coated with a material other than PVC. In an exemplary embodiment, the method includes administering to the subject a composition (e.g., an aqueous solution) comprising an anti-EGFRvIII drug using an administration system (e.g., an infusion system) that includes an intravenous (IV) bag. , less than 5% of the total volume of the IV bag is air, and the IV bag contains a composition (eg, an aqueous solution) that includes an anti-EGFRvIII drug.

As used herein, the term "administration system" is synonymous with "drug administration system" and is a device for administering a drug or therapeutic agent (e.g., an anti-EGFRvIII drug) to a subject (e.g., a human subject). , or a series of components used together. The administration system, in exemplary embodiments, includes one or more of a syringe, needle, pen, inhaler, eye dropper, catheter, container (eg, intravenous (IV) container, IV bag, vial, cartridge), and the like. In various embodiments, the administration system is an infusion system for administering a drug or therapeutic agent to a subject by infusion (eg, intravenous infusion, subcutaneous infusion). In various embodiments, the infusion system is intended for intravenous administration of a drug or therapeutic agent. Optionally, the infusion system includes an IV administration set that is used to administer infusion fluids from the container into the subject's vasculature through a needle or catheter inserted into a vein. In various embodiments, the IV administration set includes needles, catheters, tubing, flow control valves, drip chambers, infusion lines, infusion line filters, stopcocks for the IV set, fluid delivery tubing, connectors between parts of the set, injection sites, etc. It includes one or more of a functional capped side tube, an infusion pump, an infusion port, and/or a hollow spike that extends through the tube and connects to an IV bag or other infusion fluid container. In various examples, the IV administration set includes an infusion line and/or an IV bag.

In various embodiments, one or more of the components, or at least a portion of the components, of the administration system (eg, infusion system) that contacts the aqueous solution is substantially devoid of PVC or PVC plasticizer. PVC is a synthetic plastic polymer produced by polymerizing vinyl chloride monomer and is a generally hard, non-plastic material. In various examples, PVC is mixed with plasticizers to make it more flexible and/or durable. In various embodiments, the PVC is plasticized PVC. In typical examples, one or more of the components, or at least a portion of the components, of the dispensing system (e.g., infusion system) that contacts the aqueous solution is made of PVC (e.g., plasticized PVC) or PVC plasticizer (e.g., ester plasticizers, phthalate plasticizers). In various embodiments, one or more portions include a polymer other than PVC, for example, a polymer other than plasticized PVC. In various embodiments, one or more of the components, or at least a portion of the components, of the delivery system (e.g., infusion system) that contacts the aqueous solution comprises less than about 5% PVC, e.g., less than about 5% plasticized. Contains PVC. Optionally, one or more of the components, or at least a portion of the components, of the administration system (e.g., infusion system) that contacts the aqueous solution is less than about 4%, less than about 3%, less than about 2%, or about Contains less than 1% PVC, eg plasticized PVC. In exemplary embodiments, one or more of the components, or at least a portion of the components, of the administration system (e.g., infusion system) that contacts the aqueous solution is less than about 0.9%, less than about 0.8%, about less than 0.7%, less than about 0.6%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, or less than about 0.1% PVC , including, for example, plasticized PVC.

In various embodiments, one or more of the components, or at least a portion of the components, of the delivery system (e.g., infusion system) that contacts the aqueous solution is made of polyolefin elastomer (POE) or polyurethane (PUR) or ethylene vinyl acetate (EVA). )including. Optionally, one or more of the components, or at least some of the components, of the administration system (eg, the infusion system) that come into contact with the aqueous solution are composed entirely of POE, PUR or EVA. Alternatively, one or more of the components of the dispensing system (e.g., an infusion system) comprises plasticized PVC, yet the portions in contact with the aqueous solution are coated or lined with PVC or a material substantially devoid of PVC plasticizer, optionally Optionally, one or more parts in contact with the aqueous solution are coated or lined with POE, PUR or EVA. In various embodiments, at least the inside of the injection line is coated with a material other than PVC (eg, plasticized PVC), and optionally, at least the inside of the injection line is coated or lined with POE, PUR, or EVA. In various embodiments, the POE is a copolymer of either ethylene-butene or ethylene-octene. In some embodiments, the POE is an ethylene/polypropylene elastomer (EPM). Optionally, the POE is poly-4-methyl-1-pentene. In various aspects, the POE is an ENGAGE ^™ POE, an INFUSE ^™ POE, an AFFINITY ^™ GA POE, or a NORDEL ^™ EPDM POE (Dow Chemical Co., Midland, MI). Additional POEs are known in the art. For example, US Patent Application Publication No. 20180163024, McKeen, Chapter 9 "Polyolefins", Film Properties of Plastics and Elastomers ( ^4th ed.), Plastics Design L ibrary, William Andrew Applied Science Publishers, pgs. 229-272 (2017). In a typical example, a PUR is any polymer consisting of organic units linked by carbamate (urethane) bonds. Optionally, the PUR is made from di- or tri-isocyanates and polyols. In various embodiments, the isocyanate is an aromatic diisocyanate, toluene diisocyanate, or methylene diphenyl diisocyanate. In some examples, the isocyanates include 1,6-hexamethylene diisocyanate (HDI), 1-isocyanato-3-isocyanatomethyl-3,5,5-trimethyl-cyclohexane (isophorone diisocyanate, IPDI), and 4,4 '-diisocyanatodicyclohexylmethane (H ₁₂ MDI or hydrogenated MDI). In various embodiments, the polyol is a polyether polyol. In various embodiments, EVA is a copolymer of ethylene and vinyl acetate. In various embodiments, the weight percentage of vinyl acetate is from about 10% to about 40%, with the balance being ethylene. In an exemplary embodiment, the EVA contains 4% or less vinyl acetate. In alternative embodiments, the EVA comprises from about 4% to about 30% vinyl acetate, while in other embodiments, the EVA comprises greater than 60% vinyl acetate. In various embodiments, the EVA comprises about 30% to about 60% vinyl acetate. In an exemplary embodiment, the EVA is Elvax ^™ EVA copolymer resin (Dow, Midland, Mich.), DuPont Vamac ^™ EVA (DuPont, Wilmington, DE), Pre-Elec® CP 1319 EVA (Omnexus , Zurich, Switzerland), Evazote® EVA copolymer foam (Zotefoams Plc, Croydon, UK), Neolene EH (Zotefoams), or APIZERO® or APIFIVE® Trademark) EVA compound (Trinseo, Auburn Hills, MI ).

In various embodiments, one or more components, or at least a portion of the components, of a delivery system (eg, an infusion system) that contacts the aqueous solution is substantially devoid of PVC plasticizer. A plasticizer is a substance added to make a material softer and more plastic or pliable. Plasticizers can reduce the viscosity of the material or reduce the friction that occurs during handling of the material during manufacturing. Plasticizers are commonly added to PVC to improve its softness, flexibility, and/or durability. As used herein, the term "PVC plasticizer" refers to a plasticizer that is added to PVC to improve its softness, durability, and/or flexibility. Without wishing to be bound by any particular theory, the PVC plasticizer chemically reacts with one or more components of the aqueous solution, such as polysorbate 80, forming precipitates or particles in the aqueous solution. In various embodiments, one or more components, or at least a portion of the components, of a delivery system (eg, an infusion system) that contacts the aqueous solution includes less than about 5% PVC plasticizer. Optionally, one or more of the components, or at least a portion of the components, of the administration system (e.g., infusion system) that contacts the aqueous solution is less than about 4%, less than about 3%, less than about 2%, or about Contains less than 1% PVC plasticizer. In exemplary embodiments, one or more of the components, or at least a portion of the components, of the administration system (e.g., infusion system) that contacts the aqueous solution is less than about 0.9%, less than about 0.8%, about less than 0.7%, less than about 0.6%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, or less than about 0.1% PVC Contains plasticizer. In some examples, one or more components, or at least a portion of the components, of a delivery system (e.g., an infusion system) that comes into contact with an aqueous solution is, for example, bis(2-ethylhexyl) phthalate (DEHP), bis( Phthalates such as 2-propylheptyl phthalate, diisononyl phthalate, di-n-butyl phthalate, butylbenzyl phthalate, diisodecyl phthalate, dioctyl phthalate, diisooctyl phthalate, diethyl phthalate, diisobutyl phthalate, di-n-hexyl phthalate, etc. lack In alternative or additional aspects, one or more components, or at least a portion of the components, of the administration system (e.g., infusion system) that contacts the aqueous solution is a trimellitate, e.g., trioctyl trimellitate (TOTM), trimethyl Substantially lacking trimellitate, tri-(n-octyl, n-decyl) trimellitate, tri-(heptyl, nonyl) trimellitate, n-octyl trimellitate. In alternative or additional aspects, one or more components, or at least a portion of the components, of the administration system (e.g., an infusion system) that contacts the aqueous solution is an adipate, sebacate, or maleate, e.g., bis(2-ethylhexyl). ) Substantially lacking adipate, dimethyl adipate, monomethyl adipate, dioctyl adipate, dibutyl sebacate, dibutyl maleate, diisobutyl maleate.

In an exemplary embodiment, a method includes administering an aqueous solution containing an anti-EGFRvIII drug to a subject using an infusion system that includes an infusion line. In various embodiments, the injection line or portion thereof that contacts the aqueous solution is substantially devoid of PVC or PVC plasticizer, eg, contains less than about 5% PVC or PVC plasticizer. In various embodiments, the injection line includes a polymer other than PVC. In typical examples, the injection line includes POE, PUR, and/or EVA. In various embodiments, the injection line is constructed entirely of a polymer other than PVC, eg, POE, PUR, EVA. Alternatively, the injection line comprises PVC, such as plasticized PVC, and the portion of the injection line that contacts the aqueous solution is coated with a material other than PVC, such as plasticized PVC. Optionally, one or more parts in contact with the aqueous solution, for example the inner surface of the injection line, are coated with POE or PUR or EVA. Optionally, the administration system includes a container for containing the aqueous solution, at least a portion of which is substantially devoid of PVC, eg, comprises less than about 5% PVC or PVC plasticizer.

In various embodiments, the administration system includes a container for containing an aqueous solution, and when the container contains an aqueous solution, at least a portion of the container is substantially devoid of air. In various examples, when the container contains an aqueous solution, less than about 5% of the volume of the container is air. Optionally, when the container contains an aqueous solution, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the volume of the container is air. In various embodiments, at least a portion of the IV bag is substantially devoid of PVC or PVC plasticizer, and optionally, a portion of the IV bag that contacts the aqueous solution is substantially devoid of PVC or PVC plasticizer. In some examples, at least a portion of the IV bag comprises ethylene vinyl acetate (EVA) or polyolefin elastomer (POE). For example, IV bags are constructed entirely of EVA or POE.

In an exemplary embodiment, a method includes administering an aqueous solution containing an anti-EGFRvIII drug to a subject using an infusion system that includes an IV bag. In some embodiments, when the IV bag includes an aqueous solution, less than about 5% of the volume of the container is air. Optionally, when the IV bag includes an aqueous solution, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the volume of the IV bag is air. In various embodiments, at least a portion of the IV bag is substantially devoid of PVC or PVC plasticizer, and optionally, a portion of the IV bag that contacts the aqueous solution is substantially devoid of PVC or PVC plasticizer. In some examples, at least a portion of the IV bag comprises ethylene vinyl acetate (EVA) or polyolefin elastomer (POE). For example, IV bags are constructed entirely of EVA or POE.

In an exemplary embodiment, the aqueous solution is administered to the subject by intravenous (IV) infusion, optionally continuous intravenous infusion (cIVi). In a typical example, an anti-EGFRvIII drug is administered to a subject as a constant flow cIVi. In some embodiments, the aqueous solution is injected at an injection rate of about 1.5 ml/hr to about 150 ml/hr. In various examples, the aqueous solution is injected at an injection rate of about 1.5 ml/hour to about 5 mL/hour.

The infusion system, in various embodiments, further includes an infusion line filter, an intravenous (IV) bag, an infusion pump, or a combination thereof. Optionally, the infusion line filter is a polyethylsulfone (PES) filter. In various examples, the infusion pump is a CADD pump. Optionally, the infusion pump is programmable, lockable, non-elastomeric, and/or has an alarm.

Without wishing to be bound by any particular theory, the methods of the present disclosure result in reduced particulation of anti-EGFRvIII drugs during infusion, such that sufficient drug The amount and/or effective amount of the drug is provided to the subject. The quality of aqueous solutions administered to a subject in various ways is expressed by the properties of the post-administration solution, i.e., the aqueous solution containing the anti-EGFRvIII drug that has been passed through an administration system, e.g., an infusion system, collected, and then analyzed in vitro. be done. In an exemplary embodiment, the average cumulative count of subvisible particles per mL of aqueous solution administered to the subject is less than about 15. The average cumulative count of subvisible particles per mL can be determined using a HIAC™ liquid particle counter, such as ^{the HIAC™} 8011+ or HIAC ^{™ 9703+} liquid particle counter (Beckman Coulter, Indianapolis, IN). It may be measured by In various embodiments, the subvisible particles are less than about 125 μm, such as about 10 μm or about 25 μm. Optionally, the average cumulative count of subvisible particles per mL of aqueous solution administered to the subject is less than about 10 (e.g., less than about 9, less than about 8, less than about 7, less than about 6, less than about 5, about less than 4, less than about 3, less than about 2, less than about 1). In exemplary embodiments, the average cumulative count of subvisible particles per mL of solution after administration is less than 15, optionally less than about 10 (e.g., less than about 9, less than about 8, about less than 7, less than about 6, less than about 5, less than about 4, less than about 3, less than about 2, less than about 1). In an exemplary embodiment, the aqueous solution administered to the subject has less than 5 visible particles of 125 μm or larger. Optionally, the post-administration solution upon visual inspection has less than 5 visible particles of 125 μm or larger. In various embodiments, the proportion of high molecular weight (HMW) species in the aqueous solution administered to the subject is less than 5%. In various examples, the percentage of high molecular weight (HMW) species in the aqueous solution administered to the subject is less than 2%, and/or the percentage of the main peak of anti-EGFRvIII drug is greater than 95%. Optionally, the proportion of high molecular weight (HMW) species in the solution after administration is less than 5% as determined by SE-HPLC. In various instances, the percentage of high molecular weight (HMW) species in the solution after administration is less than 2%, and/or the percentage of the main peak of the anti-EGFRvIII drug is greater than 95%, as measured by SE-HPLC. be. In an exemplary embodiment, greater than 95% of the anti-EGFRvIII drug in aqueous solution before passing through the administration system (eg, infusion system) is administered to the subject. In an exemplary embodiment, the recovery of anti-EGFRvIII drug in solution after administration is greater than 95% as measured by UV-VIS spectroscopy or RP-HPLC.

Regarding the methods and kits of the present disclosure, in an exemplary embodiment, an aqueous solution containing an anti-EGFRvIII drug is suitable for administration to a subject by injection. In various embodiments, the aqueous solution is sterile and isotonic. In typical examples, the aqueous solution comprises saline, optionally from about 0.5% (v/v) to about 1.5% (v/v) saline. In various examples, the aqueous solution is about 0.5% (v/v) to about 1.4% (v/v) saline, about 0.5% (v/v) to about 1.3% (v/v) saline, about 0.5% (v/v) to about 1.2% (v/v) saline, about 0.5% (v/v) to about 1. 1% (v/v) saline, about 0.5% (v/v) to about 1.0% (v/v) saline, about 0.5% (v/v) to about 0.9% (v/v) saline, about 0.5% (v/v) to about 0.8% (v/v) saline, about 0.5% (v/v) ~0.7% (v/v) saline, ~0.5% (v/v) ~0.6% (v/v) saline, ~0.6% (v/v) v) to about 1.5% (v/v) saline, about 0.7% (v/v) to about 1.5% (v/v) saline, about 0.8% ( v/v) to about 1.5% (v/v) saline, about 0.9% (v/v) to about 1.5% (v/v) saline, about 1.0 % (v/v) to about 1.5% (v/v) saline, about 1.1% (v/v) to about 1.5% (v/v) saline, about 1 .2% (v/v) to about 1.5% (v/v) saline, about 1.3% (v/v) to about 1.5% (v/v) saline, or about 1.4% (v/v) to about 1.5% (v/v) saline.

In an exemplary embodiment, the aqueous solution includes an intravenous solution stabilizer (IVSS). IVSS is described in WO 2018/204907, which is incorporated herein by reference. In various embodiments, the IVSS is a sterile, preservative-free, colorless solution containing citric acid monohydrate, lysine hydrochloride, polysorbate 80, sodium hydroxide to adjust the pH to 7.0, and water for injection. It is a slightly yellow clear solution. In an exemplary embodiment, the IVSS comprises about 25 mM citric acid, about 1.25 M L-lysine HCl, about 0.1% (w/v) polysorbate 80 at pH 7.0. In various examples, IVSS contains citric acid monohydrate (52.5 mg), lysine hydrochloride (2283.8 mg), polysorbate 80 (10 mg), sodium hydroxide to adjust pH to 7.0, and injection Provided as a single dose in a vial containing water. In various embodiments, the aqueous solution comprises about 2% (v/v) to about 6% (v/v) IVSS, optionally about 4% (v/v) IVSS. In an exemplary embodiment, the aqueous solution comprises about 2% (v/v) to about 5.5% (v/v) IVSS, about 2% (v/v) to about 5.0% (v/v) IVSS of about 2% (v/v) to about 4.5% (v/v) IVSS of about 2% (v/v) to about 4.0% (v/v), about 2% (v/v) to about 3.5% (v/v) IVSS, about 2% (v/v) to about 3.0% (v/v) IVSS, about 2% (v/v) to about 2.5% (v/v) IVSS, about 2.5% (v/v) to about 6% IVSS, about 3.0% (v/v) to about 6% IVSS, about 3. 5% (v/v) to about 6% IVSS, about 4.0% (v/v) to about 6% IVSS, about 4.5% (v/v) to about 6% IVSS, about 5 0% (v/v) to about 6% IVSS, or about 5.5% (v/v) to about 6% IVSS. To facilitate use of the IVSS, it can be provided as prefilled vials for bedside mixing, for example, at about 10 mL per vial in prefilled vials. The target fill volume for each vial may be slightly higher to account for potential loss during clinical use (eg, target fill volume of approximately 10.6 mL per vial).

In various embodiments, the anti-EGFRvIII agent is present in the aqueous solution at a concentration of less than 75 ng/mL, optionally less than 50 ng/mL. In some examples, the anti-EGFRvIII drug is present in the aqueous solution at a concentration of about 75 ng/mL to about 500 ng/mL, optionally about 200 ng/mL to about 400 ng/mL. In exemplary embodiments, the final volume of the aqueous solution is about 100 mL to about 300 mL of solution. In representative examples, the anti-EGFRvIII drug is any one of those described herein. In certain embodiments, an anti-EGFRvIII agent described herein comprises the amino acid sequence of SEQ ID NO: 12. In certain embodiments, an anti-EGFRvIII agent described herein comprises the amino acid sequence of SEQ ID NO:13.

In various examples, the anti-EGFRvIII drug is present in an aqueous solution at a concentration ranging from 0.5 mg/ml to 20 mg/ml, preferably from 0.5 mg/ml to 10 or 5 mg/ml, the aqueous solution comprising benzyl alcohol, At least one preservative selected from chlorobutanol, metacresol, methylparaben, phenoxyethanol, propylparaben and thimerosal at a concentration effective to inhibit microbial growth, and a diluent. Optionally, the aqueous solution is (a) 0.5-200 g/ml at a pH of 6.5-7.5, or (b) 0.5-1000 g/ml at a pH of 4.0-6.0. or (c) 0.5 mg to 2 mg in the presence of a CD3 binding domain stabilizer, preferably citrate, at a pH of 4.0 to 7.5, or (d) 4.0 to 7.5, preferably The anti-EGFRvIII drug is present in a concentration range selected from the group consisting of 0.5 mg to 20 mg, preferably 0.05 mg/ml to 10 or 5 mg/ml at a pH of 4.0 to 6.0. In various examples, the preservative is present at a concentration ranging from 0.001 to 1.0% (w/v). Optionally, the preservative is present in a concentration ranging from 0.009 to 0.9% (w/v), preferably from 0.11 to 0.9% or from 0.5 to 0.75% (w/v). exists in In various embodiments, the diluent is a buffer comprising a salt selected from the group consisting of phosphate, acetate, citrate, succinate, and tartrate, and/or the buffer comprises a histidine salt. , glycine, tris-glycine, tris, or mixtures thereof. In representative examples, the diluent is a group consisting of potassium phosphate, acetic acid/sodium acetate, citric acid/sodium citrate, succinic acid/sodium succinate, tartaric acid/sodium tartrate, and histidine/histidine HCl or mixtures thereof. A buffer solution selected from Optionally, the diluent is a buffer present at a concentration in the range 0.1-150mM, preferably in the range 0.25-50mM. In various embodiments, the diluent is a buffer solution that optionally includes citrate at a concentration ranging from 0.25 to 50 mM. In some embodiments, the pH of the composition is in the range 4.0-8.0, preferably pH 4.0-5.0 or 6.0-7.5, preferably pH 7.0. In an exemplary embodiment, the aqueous solution contains one or more excipients selected from the group consisting of sucrose, trehalose, mannitol, sorbitol, arginine, lysine, polysorbate 20, polysorbate 80, poloxamer 188, pluronics, and combinations thereof. Including further. Optionally, the aqueous solution comprises polysorbate, preferably polysorbate 80, and/or lysine HCl. In a typical example, the aqueous solution is free of polysorbate, preferably polysorbate 80, and/or lysine HCl when the concentration of anti-EGFRvIII drug is at least 10, 15 or 20 μg/ml, preferably 15 μg/ml. In various embodiments, the aqueous solution comprises at least 0.25 mM citrate, at least 0.0125 mM lysine, and/or at least 0.001% polysorbate 80 at a pH of 6.5-7.5. In various embodiments, the anti-EGFRvIII agent is administered at an initial dose of about 15 μg/day to about 12000 μg/day for at least 14 days. In various examples, the anti-EGFRvIII drug is administered at an initial dose of about 1500 μg/day to about 6000 μg/day for at least 14 days. In a typical example, the anti-EGFRvIII drug is administered at an initial dose of about 3000 μg/day to about 6000 μg/day for at least 14 days. In an exemplary embodiment, the anti-EGFRvIII drug is administered at an initial dose of about 15 μg/day to about 12000 μg/day for at least 28 days. In various embodiments, the anti-EGFRvIII agent is administered at an initial dose of about 1500 μg/day to about 6000 μg/day for at least 28 days. The anti-EGFRvIII drug is administered at an initial dose of about 3000 μg/day to about 6000 μg/day for at least 28 days in various examples. In typical examples, the anti-EGFRvIII drug is administered in a 14 day on/14 day off cycle, or a 28 day on/14 day off cycle. The anti-EGFRvIII drug is administered to the subject in an exemplary embodiment at a dose of 3000 μg/day to 6000 μg/day on a 14 day on/14 day off cycle or a 28 day on/14 day off cycle.

In an exemplary embodiment of the present disclosure, the subject is a mammal of the order Rodentia, such as, but not limited to, mice and hamsters, and mammals of the order Logomorpha, such as rabbits. animals, mammals of the order Carnivora, including Felidae (cats) and Canidae (dogs), mammals of the order Artiodactyla, including Bovidae (cows) and Boaridae (pigs), or horses Mammals, including mammals of the order Persodactyla, including the family (horses). In some embodiments, the mammal is a mammal of the order Primates, Ceboid, or Simoid (monkeys), or a mammal of the order Anthropidae (humans and apes). In some embodiments, the mammal is a human. In various embodiments, the subject has a neoplastic disease, such as any one of those described herein. The terms "patient," "subject," or "mammal," as used herein, refer to any "patient," "subject," or "mammal," including humans, cows, horses, dogs, and cats. Point. In one embodiment of the invention, the mammal is a human. In various examples, the subject is an adult human, eg, 18 years of age or older.

In various embodiments, the subject has a neoplastic disease and the method treats the neoplastic disease. As used herein, the term "neoplastic disease" refers to any condition that causes tumor growth. In an exemplary embodiment, the tumor is a benign tumor. In an exemplary embodiment, the tumor is a malignant tumor. In various embodiments, the neoplastic disease is cancer. Cancer in its various forms includes acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, and cancer of the eyeball. cancer of the intrahepatic bile duct, joints, neck, gallbladder or pleura, nose, nasal cavity or middle ear, oral cavity cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid Cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumor, Hodgkin lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma cancer, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental, mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer ( For example, renal cell carcinoma (RCC)), small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, or bladder cancer. In certain aspects, the cancer is head and neck cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, pancreatic cancer, gastrointestinal cancer, gastric cancer, breast cancer, endometrial cancer, Colorectal cancer, hepatocellular carcinoma, glioblastoma, bladder cancer, lung cancer, such as non-small cell lung cancer (NSCLC) or bronchioloalveolar carcinoma. In certain embodiments, the tumor is non-small cell lung cancer (NSCLC), head and neck cancer, kidney cancer, triple negative breast cancer, or gastric cancer. In an exemplary embodiment, the subject has a tumor (e.g., a solid tumor, hematologic malignancy, or lymphoid malignancy), and the pharmaceutical composition is administered to the subject in an amount effective to treat the subject's tumor. Ru. In other exemplary embodiments, the tumor is non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), head and neck cancer, kidney cancer, breast cancer, melanoma, ovarian cancer, liver cancer, pancreatic cancer. cancer, colon cancer, prostate cancer, gastric cancer, lymphoma, or leukemia, and the pharmaceutical composition is administered to the subject in an amount effective to treat the subject's tumor.

The terms "cancer" and "cancerous" as used herein refer to or describe the physiological condition in mammals that is typically characterized by uncontrolled cell growth. It is something. Examples of cancer include, but are not limited to, carcinoma, lymphoma, sarcoma, blastoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, lung cancer, pancreatic cancer, cervical cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon carcinoma, and head and neck cancer, ovarian cancer. cancer, and endometrial cancer.

In various embodiments, the neoplastic disease is cancer or a tumor. Optionally, the cancer or tumor is an EGFRvIII positive cancer or tumor. In a typical example, the cancer is a squamous cell tumor, such as non-small cell lung cancer (NSCLC). In exemplary embodiments, the cancer is glioblastoma or malignant glioma.

The term "treatment" includes prophylactic and/or therapeutic treatment. Treatment is considered prophylactic treatment when administered before clinical signs of a disease condition. Therapeutic treatment includes, for example, improving or reducing the severity of the disease or shortening the duration of the disease. The term "treat", as well as its related terms, does not necessarily mean 100% or complete treatment. Rather, there are varying degrees of treatment that are recognized by those skilled in the art as having potential benefit or therapeutic effect. In this regard, the methods of treating neoplastic diseases of the present disclosure can provide any amount or level of treatment. Additionally, treatment provided by the methods of the present disclosure may include treatment of one or more conditions or symptoms or signs of the neoplastic disease being treated. Treatments provided by the methods of the present disclosure may also include slowing the progression of neoplastic diseases. For example, the methods may enhance T cell activity or immune responses to neoplastic diseases; reduce tumor or cancer growth or tumor burden; reduce tumor cell metastasis; Neoplastic diseases can be treated, such as by increasing cell death or by increasing tumor regression. In accordance with the above, provided herein are methods of reducing tumor growth or tumor burden or increasing tumor regression in a subject. In an exemplary embodiment, the method includes administering to the subject an anti-EGFRvIII drug. The terms "treat," "treating," and "treatment" as used herein refer to therapy including, but not limited to, curative therapy, prophylactic therapy, and prophylactic therapy. Preventive treatment generally consists of either preventing the development of the disorder altogether in an individual or delaying the onset of the disorder at a preclinically evident stage.

In various embodiments, the method prevents the onset or recurrence of the neoplastic disease by at least 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 15 days, 30 days, 2 months, 3 months, 4 months. , treatment with the purpose of delaying treatment by 6 months, 1 year, 2 years, 3 years, or 4 years or more. In various embodiments, the method treats the subject for the purpose of increasing survival. In an exemplary aspect, the methods of the present disclosure provide treatment for the purpose of delaying the occurrence or development of metastases. In various examples, the methods provide treatment for the purpose of delaying the development or development of new metastases. Accordingly, provided herein are methods of delaying the occurrence or development of metastases in a subject suffering from cancer. In an exemplary embodiment, the method includes administering to the subject an anti-EGFRvIII drug.

Typically, the treatment provided can be described in terms of or supported by data obtained from a clinical trial, and the endpoints of the trial include progression-free survival (PFS), progression-free survival (PFS), Overall survival (OS) or time to deterioration of general status according to the East Coast Cancer Group (ECOG). In various aspects, the present disclosure provides methods of prolonging the time to deterioration of PFS, OS, or ECOG performance status in a subject suffering from a neoplastic disease. As used herein, the term "progression-free survival" or "PFS" refers to the period of time that a treated patient will experience without the cancer worsening (by whatever metric used to measure worsening). It means time to do something. The term "overall survival" means how long a patient survives after treatment. ECOG performance status assesses the patient's disease (e.g., how the disease progresses/regresses, how the disease affects the patient's ability to perform daily activities) and determines appropriate treatment and prognosis. grade or score based on a scale used by physicians and researchers to ECOG general status is determined according to the following criteria.

5. Kits and Articles of Manufacture The present disclosure provides kits. In an exemplary embodiment, the kit includes an administration system and an anti-EGFRvIII agent, one or more of the components of the administration system, or portions thereof, that contact the aqueous solution are substantially devoid of PVC or PVC plasticizer, and /or substantially devoid of air. In various embodiments, the administration system is any of those described herein, including, but not limited to, an infusion system that includes an infusion line. In various embodiments, one or more of the components of the administration system (eg, the injection system), or portions thereof, that contact the aqueous solution are substantially devoid of PVC or PVC plasticizer. In various embodiments, one or more portions include a polymer other than PVC, eg, plasticized PVC. In typical examples, one or more portions include polyolefin elastomer (POE) or polyurethane (PUR) or ethylene vinyl acetate (EVA). Optionally, one or more portions are composed entirely of POE, PUR, or EVA. Alternatively, one or more parts comprise PVC, for example PVC with a plasticizer, and the parts in contact with the aqueous solution are coated with a material other than PVC. Optionally, one or more parts in contact with the aqueous solution are coated with POE or PUR, or EVA. In various embodiments, the injection line or portion thereof that contacts the aqueous solution is substantially devoid of PVC or PVC plasticizer. In various embodiments, the injection line includes a polymer other than PVC. In typical examples, the injection line includes POE and/or PUR and/or EVA. In various embodiments, the injection line is constructed entirely of a polymer other than PVC, for example, the injection line is constructed entirely of POE, PUR, or EVA. Alternatively, the injection line comprises PVC, yet the portion of the injection line that contacts the aqueous solution is coated with a material other than PVC, for example coated with POE, PUR, or EVA. Optionally, one or more parts in contact with the aqueous solution, for example the inner surface of the injection line, are coated with POE or PUR or EVA. Optionally, the dosing system includes a container for containing the aqueous solution, at least a portion of which is substantially free of PVC or PVC plasticizer.

In various embodiments, the administration system includes a container for containing an aqueous solution, and when the container contains an aqueous solution, at least a portion of the container is substantially devoid of air. In various examples, when the container contains an aqueous solution, less than about 5% of the volume of the container is air. Optionally, when the container contains an aqueous solution, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the volume of the container is air. In various embodiments, at least a portion of the IV bag is substantially devoid of PVC or PVC plasticizer, and optionally, a portion of the IV bag that contacts the aqueous solution is substantially devoid of PVC or PVC plasticizer. In some examples, at least a portion of the IV bag comprises ethylene vinyl acetate (EVA) or polyolefin elastomer (POE). For example, IV bags are constructed entirely of EVA or POE. In an exemplary embodiment, a method includes administering an aqueous solution containing an anti-EGFRvIII drug to a subject using an infusion system that includes an IV bag. In some embodiments, when the IV bag includes an aqueous solution, less than about 5% of the volume of the container is air. Optionally, when the IV bag includes an aqueous solution, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the volume of the IV bag is air. In various embodiments, at least a portion of the IV bag is substantially devoid of PVC or PVC plasticizer, and optionally, a portion of the IV bag that contacts the aqueous solution is substantially devoid of PVC or PVC plasticizer. In some examples, at least a portion of the IV bag comprises ethylene vinyl acetate (EVA) or polyolefin elastomer (POE). For example, IV bags are constructed entirely of EVA or POE.

In various embodiments, the aqueous solution of exemplary embodiments comprising an anti-EGFRvIII agent comprises saline, optionally about 0.5% to about 1.5% (v/v) saline. In various examples, the aqueous solution is about 0.5% to about 1.4% (v/v) saline, about 0.5% to about 1.3% (v/v) saline, about 0.5% to about 1.2% (v/v) saline, about 0.5% to about 1.1% (v/v) saline, about 0.5% to about 1 .0% (v/v) saline, about 0.5% to about 0.9% (v/v) saline, about 0.5% to about 0.8% (v/v) of saline, about 0.5% to about 0.7% (v/v) saline, about 0.5% to about 0.6% (v/v) saline, about 0. 6% to about 1.5% (v/v) saline, about 0.7% to about 1.5% (v/v) saline, about 0.8% to about 1.5% (v/v) saline, about 0.9% to about 1.5% (v/v) saline, about 1.0% to about 1.5% (v/v) saline Water, about 1.1% to about 1.5% (v/v) saline, about 1.2% to about 1.5% (v/v) saline, about 1.3% to About 1.5% (v/v) saline, or about 1.4% to about 1.5% (v/v) saline. Optionally, the aqueous solution comprises about 2% (v/v) to about 6% (v/v) intravenous saline solution (IVSS), optionally about 4% (v/v) IVSS. In an exemplary embodiment, the aqueous solution comprises about 2% (v/v) to about 5.5% (v/v) IVSS, about 2% (v/v) to about 5.0% (v/v) IVSS of about 2% (v/v) to about 4.5% (v/v) IVSS of about 2% (v/v) to about 4.0% (v/v), about 2% (v/v) to about 3.5% (v/v) IVSS, about 2% (v/v) to about 3.0% (v/v) IVSS, about 2% (v/v) to about 2.5% (v/v) IVSS, about 2.5% (v/v) to about 6% IVSS, about 3.0% (v/v) to about 6% IVSS, about 3. 5% (v/v) to about 6% IVSS, about 4.0% (v/v) to about 6% IVSS, about 4.5% (v/v) to about 6% IVSS, about 5 0% (v/v) to about 6% IVSS, or about 5.5% (v/v) to about 6% IVSS. In various embodiments, the anti-EGFRvIII agent is present in the aqueous solution at a concentration of less than 75 ng/mL, optionally less than 50 ng/mL. In some examples, the anti-EGFRvIII drug is present in the aqueous solution at a concentration of about 75 ng/mL to about 500 ng/mL, optionally about 200 ng/mL to about 400 ng/mL. In exemplary embodiments, the final volume of the aqueous solution is about 100 mL to about 300 mL of solution. In representative examples, the anti-EGFRvIII drug is any one of those described herein. In certain embodiments, an anti-EGFRvIII agent described herein comprises the amino acid sequence of SEQ ID NO: 12. In certain embodiments, an anti-EGFRvIII agent described herein comprises the amino acid sequence of SEQ ID NO:13.

The present disclosure provides an article of manufacture. In an exemplary embodiment, the article of manufacture includes a container containing an aqueous solution comprising an anti-EGFRvIII drug, the container being substantially devoid of air. In various examples, when the container contains an aqueous solution, less than about 5% of the volume of the container is air. Optionally, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the volume of the container is air. In various embodiments, the container is an IV bag, and at least a portion of the IV bag is substantially free of PVC or PVC plasticizer. In some examples, at least a portion of the IV bag comprises ethylene vinyl acetate (EVA) or polyolefin elastomer (POE). For example, the IV bag may be constructed entirely of EVA or POE, or the inside of the IV bag may be lined or coated with EVA or POE. In typical examples, the aqueous solutions of exemplary embodiments containing anti-EGFRvIII agents include saline, optionally about 0.5% to about 1.5% (v/v) saline. In various examples, the aqueous solution is about 0.5% to about 1.4% (v/v) saline, about 0.5% to about 1.3% (v/v) saline, about 0.5% to about 1.2% (v/v) saline, about 0.5% to about 1.1% (v/v) saline, about 0.5% to about 1 .0% (v/v) saline, about 0.5% to about 0.9% (v/v) saline, about 0.5% to about 0.8% (v/v) of saline, about 0.5% to about 0.7% (v/v) saline, about 0.5% to about 0.6% (v/v) saline, about 0. 6% to about 1.5% (v/v) saline, about 0.7% to about 1.5% (v/v) saline, about 0.8% to about 1.5% (v/v) saline, about 0.9% to about 1.5% (v/v) saline, about 1.0% to about 1.5% (v/v) saline Water, about 1.1% to about 1.5% (v/v) saline, about 1.2% to about 1.5% (v/v) saline, about 1.3% to About 1.5% (v/v) saline, or about 1.4% to about 1.5% (v/v) saline. Optionally, the aqueous solution comprises about 2% (v/v) to about 6% (v/v) intravenous saline solution (IVSS), optionally about 4% (v/v) IVSS. In an exemplary embodiment, the aqueous solution comprises about 2% (v/v) to about 5.5% (v/v) IVSS, about 2% (v/v) to about 5.0% (v/v) IVSS of about 2% (v/v) to about 4.5% (v/v) IVSS of about 2% (v/v) to about 4.0% (v/v), about 2% (v/v) to about 3.5% (v/v) IVSS, about 2% (v/v) to about 3.0% (v/v) IVSS, about 2% (v/v) to about 2.5% (v/v) IVSS, about 2.5% (v/v) to about 6% IVSS, about 3.0% (v/v) to about 6% IVSS, about 3. 5% (v/v) to about 6% IVSS, about 4.0% (v/v) to about 6% IVSS, about 4.5% (v/v) to about 6% IVSS, about 5 0% (v/v) to about 6% IVSS, or about 5.5% (v/v) to about 6% IVSS. In various embodiments, the anti-EGFRvIII agent is present in the aqueous solution at a concentration of less than 75 ng/mL, optionally less than 50 ng/mL. In some examples, the anti-EGFRvIII drug is present in the aqueous solution at a concentration of about 75 ng/mL to about 500 ng/mL, optionally about 200 ng/mL to about 400 ng/mL. In exemplary embodiments, the final volume of the aqueous solution is about 100 mL to about 300 mL of solution. In representative examples, the anti-EGFRvIII drug is any one of those described herein. In certain embodiments, an anti-EGFRvIII agent described herein comprises the amino acid sequence of SEQ ID NO: 12. In certain embodiments, an anti-EGFRvIII agent described herein comprises the amino acid sequence of SEQ ID NO:13.

In an exemplary aspect, a kit of the present disclosure includes an IV bag prefilled with an aqueous solution including saline and/or IVSS and a container containing an anti-EGFRvIII drug. Optionally, the anti-EGFRvIII drug is lyophilized. In an alternative embodiment, the container contains the anti-EGFRvIII drug in an aqueous solution comprising saline and/or IVSS.

The following examples are presented merely to illustrate the invention and are not intended to limit its scope in any way.

The following example provides an evaluation of AMG 596 infusion solution held in an IV bag and then pumped through the infusion line. Examples have shown that when AMG 596 is pumped through a polyvinyl chloride (PVC) injection line, the molecule becomes particulate and AMG 596 is not compatible with the PVC injection line. However, when injection lines lined with either polyethylene (PE) or polyurethane (PUR) were subsequently tested for compatibility with AMG 596, no protein particulation was observed. Additionally, no incompatibilities caused by contact of the product with IV bags constructed of ethylene vinyl acetate (EVA) or polyolefins were observed. For all IV bag and infusion line combinations tested, all quality attributes evaluated were at acceptable levels, indicating compliance, with the exception of visible and subvisible particle levels. When an injection line constructed of PVC was used in the compliance evaluation, the solution taken after the injection, but not before, was extremely turbid and contained unacceptable levels of visible particles indicative of protein aggregation. The solution taken directly from the IV bag prior to infusion was clear, making it conclusive that pumping through the infusion line constructed of PVC was responsible for protein aggregation and clouding of the infusion solution. Ta. However, when replacing the PVC injection line with either a PE or PUR injection line, no visible particle generation was observed, and all other quality attributes evaluated showed that the AMG 596 injection solution It was shown that it was compatible with the line. Therefore, the PVC line was identified as a cIVi component that is not particularly suitable for injecting AMG 596.

Example 1
This example describes short-term testing of AMG596 infusion solution in an IV bag.

The AMG 596 formulation (DP) is mixed in an IV bag with saline and intravenous solution stabilizer (IVSS) to form an infusion solution. This solution is intended for infusion into the patient by pumping it into the patient through an infusion line with an infusion pump. Initial evaluation was performed in which AMG 596 infusion solution was held in an IV bag (250 mL EVA IV bag, MediBag, Beavercreek, OH) and then pumped through the infusion line using a CADD administration set (100 mL). In this study, the infusion solution contained saline, AMG 596 (either 33 μg/mL or 330 μg/mL) with or without ±4% (v/v) IVSS. . The final volume of the IV bag was 100 mL. The bag was stirred at 60 rpm for 48 hours at 25°C. The infusion solution in the IV bag was then evaluated for particle counts by HIAC, changes in the percentage of oligomers and/or fragments by SEC, and protein recovery by UV-Vis spectroscopy.

FIG. 1 shows the results of the initial evaluation. As shown in this figure, the infusion solution within the IV bag showed heavy particulation after stirring. The concentration of AMG 596 decreased to 67% of the initial concentration at 24 hours.

Given protein loss and severe particulation, alternative IV bags were evaluated. In this study, five additional IV bags consisting of EVA or POE were evaluated along with the MediBag IV bag used in the initial evaluation. Among the IV bags, Exactamix EVA IV bag (250 mL, Baxter Healthcare Corp., Deerfield, IL), Secure EVA IV bag (50 mL, Medisca, Quebec, Canada), and ASI EVA IV bag (100mL, Advanced Scientific, Inc. ., Millersburg, PA), B Braun Excel POE IV Bag (250 mL, B. Braun Medical, Inc., Bethlehem, PA), and B Braun PAB® POE IV Bag (150 mL, B. Braun Me dical, Inc., Bethlehem, PA). Each bag was filled with a total volume of 100 mL of infusion solution containing AMG 596 (either 33 μg/mL or 330 μg/mL) in IVSS and saline, with or without air. The bag was stirred at 60 rpm for 48 hours at 25°C. The infusion solution in the IV bag was then evaluated for particle counts by HIAC, changes in the percentage of oligomers and/or fragments by SEC, and protein recovery by UV-Vis spectroscopy.

Exemplary results are shown in Figures 2A and 2B. As shown in these figures, infusion solutions in IV bags containing air consistently exhibited cloudiness and severe particulation after agitation compared to infusion solutions in IV bags without air. Ta. Particle counts were determined by HIAC for particles of different sizes. Figure 2C shows the number of particle counts per mL for particles with a size of 2 μm or more and 5 μm or more, whereas Figure 2D shows the number of particle counts per mL for particles with a size of 10 μm or more and 25 μm or more. . These results show that the ASI IV bag (red bars in Figures 2C and 2D) had significantly higher particle counts than the other IV bags. MediBag had the lowest number of particles ≧10 μm and the lowest number of particles ≧25 μm. Protein recovery was determined to be 100% when air was purged from the IV bag (Figure 2E). Size exclusion chromatography demonstrated that the presence or absence of air did not change the level of dimer in any of the IV bags tested after stirring at 60 rpm for 24 hours at 25°C. In the case of the Exactamix IV bag, an additional "leachable" peak appeared in the retention time of small molecules in the chromatogram.

In subsequent experiments, an in-line filter was placed between the solution set and the catheter to determine whether the filter was able to adsorb AMG 596. Infusion solutions containing AMG 596 (either 33 μg/mL or 330 μg/mL) were pumped through the infusion line using a CADD cassette (100 mL) equipped with a PES filter and protein recovery was measured. No reduction in AMG 596 could be detected using an in-line filter.

These results suggest that AMG 596 infusion solution is compatible with IV bags constructed from ethylene vinyl acetate (EVA) or polyolefin (POE).

Example 2
This example describes the materials and methods used to conduct the experiment of Example 1.

Materials - The following materials were used: CADD® administration set (274 cm (108 in. ), 3.3 mL) (Smiths Medical, St. Paul, MN); EVA IV bag, 100-300 mL, phthalate-free (e.g., DEHP-free) (ICU Medical, San Clemente, CA); CADD infusion pump; mPolyolefin IV Bag (150 mL), B Braun, PAB Partial Additive Bag, S5904-52; Intravia IV Bag, 500 mL (p/n 2J8003); Clear infusion set lined with Sapphire primary polyethylene (p/n 16422-01);QCore Medical, Sapphire infusion pump (ref# 15031-000-0028); Saline: Hospira, NDC: 0409-4888-06; Filter: B Braun, Super membrane 0.2m air removal filter, p/n 473036.

I performed the following method.

Chemical Modification of Molecules by Cation Exchange (CEX) Chromatography This technique was implemented to determine the purity and charge variant distribution of AMG 596. Charge variants of AMG 596 were separated by cation exchange chromatography using a cation exchange analytical column and then eluted from the column using a mobile phase gradient of increasing ionic strength. Proteins with less positive surface charge eluted earlier than proteins with more positive surface charge. The eluted charge variants were detected by UV detection and integrated, and the results for the main, acidic, and basic peaks were reported as a percentage of the total peak area.

Oligomerization or Fragmentation by Size Exclusion Chromatography (SEC) This method was carried out to determine the purity of AMG 596. SE-UHPLC uses size exclusion ultrafast analytical columns to separate proteins in solution based on their hydrodynamic volume. High molecular weight (aggregate peaks) eluted earlier than monomer or lower molecular weight peaks. Components were eluted at uniform concentrations, detected by UV detection, integrated, and results reported as a percentage of the relative peak area of the high molecular weight peak, the main peak (monomer), and the low molecular weight peak, if present.

pH of solution
The pH of the sample was measured using a calibrated pH meter.

Subvisible Particle Level by Light Obscuration (HIAC) This procedure measures the number of particles ≧10 μm and ≧25 μm per container. This test was conducted to count subvisible particles within a specific size range. The equipment used was an electronic liquid particle counting system using a light shielding sensor with an appropriate sample delivery device. Four aliquots (>1 mL each) were degassed by vacuum from a pooled solution with a total volume of ≥5 mL and analyzed; the data for the first aliquot was discarded and the average of the remaining 3 measurements was calculated based on the average of the remaining 3 measurements. The number of particles was calculated. Results were reported as number of particles per container with particle sizes ≧10 μm and ≧25 μm. In addition, particles with sizes ≧2 μm and ≧5 μm were observed per container.

Visible Particle Levels by Visual Inspection Samples were visually inspected using a light that illuminated the samples in a visual inspection booth. The number of particles with size ≧125 μm was recorded.

Protein Recovery Assay: UV-Vis Spectrophotometry Protein concentration of AMG 596 samples was measured using UV-Vis spectroscopy for 0.2 mg/mL AMG 596 IV bags. The absorbance of the sample was measured and the protein concentration was calculated using the following formula:
[(A ₂₈₀ −A ₃₅₀ )/2.0] (the attenuation coefficient of AMG 596 is 2.0 mg ⁻¹ *cm ⁻¹ *mL).

Protein Recovery Assay: Reversed Phase Titration Method The protein concentration of the AMG596 sample with a concentration below the detection limit of UV-visible spectroscopy was measured using reverse phase chromatography for a 90 ng/mL IV bag. Separation of AMG 596 from other impurities was achieved using a C8 column. AMG 596 was eluted from the column using a mixture of aqueous and organic solvents. Protein concentration was determined by measuring the peak area of eluted AMG 596 and standard curve and linear regression analysis, and the concentration of the sample was calculated.

Example 3
This example describes long-term testing of AMG596 infusion solution in an IV bag.

The evaluation was set up to mimic how the product would be prepared and administered to patients in the clinic. The final formulation consisted of 4% (v/v) IVSS, 0.9% (v/v) saline, and AMG 596 formulation (DP) of either 0.2 mg/mL or 90 ng/mL. IV bags were prepared with solutions having a volume of either 126 mL or 150 mL, respectively. The IV bags were then held at 2-8° C. for 10 days to accommodate the 7-day maximum holding time of the prepared IV bags. The IV bag was then placed on a shaker (60 rpm) at a controlled temperature of 30° C. for 5 days. This part of the experiment was designed to simulate keeping the patient on the IV bag for 5 days to accommodate the maximum continuous dosing period of 4 days. Finally, pump and collect the AMG 596 infusion solution through the infusion line to simulate injecting the solution into the patient.

Samples of the infusion solution were taken at the following times: after mixing in the IV bag (t0) and after 10 days of holding at 2-8°C, the solution obtained directly from the IV bag (before injection) and through the infusion line. After a further 5 days of continuous stirring of both the pumped solution (after injection) at 30°C. The sample taken at t=0 was obtained directly from the IV bag with a syringe. The 15-day samples were pumped at either 5 mL/hour or 125 mL/hour using a CADD pump through an in-line filter that included a PVC injection line with an in-line filter. Control samples contained saline and IVSS without any protein.

These samples were analyzed and evaluated for properties to determine the quality and stability of AMG 596 protein. Properties analyzed included chemical modification of the molecule by cation exchange (CEX) chromatography, oligomerization or fragmentation by size exclusion chromatography (SEC), pH of the solution, level of subvisible particles by light shielding (HIAC), and visual inspection. Protein recovery was by UV-Vis spectrophotometry for the 0.2 mg/mL AMG 596 IV bag or reverse phase titration for the 90 ng/mL IV bag.

An overview of the experiment is summarized in Figure 3A, and details of day 1 IV bag preparation are summarized in Figure 3B.

While the control sample was clear like water and the t=0 sample containing 0.2 mg/mL AMG 256 was clear, the sample containing 0.2 mg/mL AMG 256 had a significant amount contained visible particles. Also, the 15 day sample containing 90 ng/mL AMG 256 was slightly cloudy.

We repeated the above experiment simply to evaluate the effect of the injection line, but without including the in-line filter. The results are shown in Figures 4A and 4B. As shown in these figures, removing the filter had no effect on particulation, suggesting that the injection solution was not compatible with the PVC injection line.

To evaluate the effect of infusion rate, the above experiments were performed with an infusion rate of either 5 mL/hour or 125 mL/hour. The results are shown in Figures 5A and 5B. As shown in these figures, injection rate did not affect particulation.

These results confirm that the molecule becomes particulate when AMG 596 is pumped through a polyvinyl chloride (PVC) injection line and clearly demonstrate that AMG 596 is not compatible with PVC injection lines. became. Visible particles were present with and without in-line filters, and sub-visible particles were higher than acceptable levels with and without in-line filters. Note that no significant levels of protein were adsorbed to the IV bag, infusion line, or in-line filter. Recovery of AMG 596 was within acceptable limits as determined by Biacore reverse phase chromatography or UV-visible spectrophotometry. Also, injection of the injection solution through the injection system did not result in increased levels of soluble HMW species as measured by SE-UPLC.

Example 4
This example demonstrates testing to verify the compatibility of AMG 256 injection solution with non-PVC injection lines and in-line filters.

The test was conducted substantially as described in Example 3, except that an injection line lined with polyurethane (PUR) and equipped with a 0.2 μm polyethersulfone (PES) filter was used. carried out.

Recovery rates and SE-HPLC results are shown in Tables 1 and 2, respectively.

During visual inspection, no visible particles (greater than 125 μm) were observed. Subvisible particle analysis by HIAC revealed very low particle counts (Table 3).

These results demonstrate that the use of polyethylene (PE) or polyurethane (PUR) lined injection lines in the injection system for injection solutions containing AMG 596 does not cause protein particulation. It suggests.

All references, including publications, patent applications, and patents, cited herein are individually and specifically incorporated by reference as if each reference were incorporated by reference and herein incorporated by reference in its entirety. Incorporated herein by reference to the same extent as if indicated.

The use of the terms "a" and "an" and "the" and similar referents in the description of the present disclosure (and in particular in the claims that follow) are used herein Unless the text indicates otherwise or the context clearly contradicts, the singular and the plural shall be construed as including both the singular and the plural. The terms "comprising", "having", "including" and "containing" include the named element but exclude other elements, unless specified otherwise. (i.e., meaning "including, but not limited to") and should be construed as a non-limiting term.

The recitation of ranges of values herein, unless otherwise indicated herein, is intended solely to serve as shorthand to refer individually to each separate value and each endpoint within the range. and each separate value and endpoint is incorporated herein as if individually recited herein.

All methods described herein can be performed in any suitable order, unless indicated otherwise herein or clearly contradicted by context. Any example or use of exemplary language (e.g., "etc.") provided herein is intended solely to make the disclosure more clear and, unless otherwise claimed, is intended to clarify the disclosure. It does not impose any limitations on the scope. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

Preferred embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Variations on these preferred embodiments will be apparent to those skilled in the art upon reading the above description. The inventors expect those skilled in the art to use such variations as appropriate, and the inventors do not intend to use the present disclosure in other ways than those specifically described herein. is intended to be implemented. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, any combination of the elements described above is encompassed by this disclosure in all possible variations thereof, unless indicated otherwise herein or clearly contradicted by context.

Claims (31)

A method of treating an EGFRvIII-positive cancer or tumor in a subject, the method comprising administering an aqueous solution containing an anti-EGFRvIII drug to the subject using an infusion system that includes an infusion line, the infusion line comprising a polychloride Substantially devoid of vinyl (PVC) or PVC plasticizer, the anti-EGFRvIII drug comprises a dual binding domain that binds to human EGFRvIII and macaque EGFRvIII, and a second binding domain that binds to human CD3. A method wherein the specific antigen binding polypeptide is a specific antigen binding polypeptide. 2. The method of claim 1, wherein the injection line comprises polyolefin elastomer (POE), polyurethane (PUR), or ethylene vinyl acetate (EVA). 3. The method of claim 1 or 2, wherein the average cumulative count of subvisible particles per mL of solution after injection is less than 15 as measured by a submerged particle counter. 4. The method of claim 3, wherein the average cumulative count of subvisible particles per mL of solution after injection is less than 10, optionally less than 1, as measured by a submerged particle counter. 5. A method according to any one of claims 1 to 4, wherein upon visual inspection, the injected solution has less than 5 visible particles of 125 μm or larger. A method according to any one of claims 1 to 5, wherein the proportion of high molecular weight (HMW) species in the solution after injection is less than 5% as determined by SE-HPLC. The proportion of HMW species in the solution after injection is less than 2% and/or the proportion of the main peak of the anti-EGFRvIII drug is greater than 95%, as measured by SE-HPLC. 6. The method according to any one of 6. The method according to any one of claims 1 to 7, wherein the recovery of the anti-EGFRvIII drug in the solution after the injection is greater than 95% as determined by UV-VIS spectroscopy or RP-HPLC. . 9. The method of any one of claims 1 to 8, wherein the aqueous solution is administered to the subject by continuous intravenous infusion (cIVi). Any of claims 1 to 9, wherein the anti-EGFRvIII drug is administered to the subject on a 14 day on/14 day off cycle or a 28 day on/14 day off cycle at a dose of 3000 μg/day to 6000 μg/day. The method described in paragraph (1). 11. The method of any one of claims 1-10, wherein the infusion system further comprises an intravenous (IV) bag, an infusion pump, an infusion line filter, or a combination thereof. 12. The method of claim 11, wherein the infusion line filter is a polyethyl sulfone (PES) filter. 13. The method of claim 11 or 12, wherein the IV bag comprises ethylene vinyl acetate (EVA) or polyolefin elastomer (POE). 14. The method of any one of claims 11-13, wherein the IV bag comprises the aqueous solution containing the anti-EGFRvIII drug and air, and less than 5% of the total volume of the IV bag is air. 15. A method according to any one of claims 11 to 14, wherein the infusion pump is a CADD pump. 16. A method according to any one of claims 11 to 15, wherein the infusion pump is programmable, lockable, non-elastomeric and/or has an alarm. 17. The method of any one of claims 1-16, wherein the aqueous solution is injected at an injection rate of about 1.5 ml/hour to about 150 mL/hour, optionally 5 mL/hour or 125 mL/hour. The method according to any one of claims 1 to 17, wherein the cancer is glioblastoma or malignant glioma. 19. The method of any one of claims 1-18, wherein the aqueous solution comprises saline, optionally from about 0.5% to about 1.5% (v/v) saline. Claim 1, wherein the solution comprises about 2% (v/v) to about 6% (v/v) intravenous saline solution (IVSS), optionally about 4% (v/v) IVSS. 20. The method according to any one of 19 to 19. 21. The method of any one of claims 1 to 20, wherein the anti-EGFRvIII drug is present in the aqueous solution at a concentration of less than 75 ng/mL, optionally less than 50 ng/mL. 22. The method of claim 21, wherein the anti-EGFRvIII drug is present in the solution at a concentration of about 75 ng/mL to about 500 ng/mL, optionally about 200 ng/mL to about 400 ng/mL. 23. The method of any one of claims 1-22, wherein the final volume of the aqueous solution is about 100 mL to about 300 mL of solution. The EGFRvIII binding domain comprises (a) (i) VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 3; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 4; and (iii) ) A heavy chain variable region (VH) comprising CDR-H3 comprising the amino acid sequence of SEQ ID NO: 5; (b) (i) VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 6; ( ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 7; and (iii) a light chain variable region (VL) comprising CDR-L3 comprising the amino acid sequence of SEQ ID NO: 8;
The CD3 binding domain comprises (a) (i) VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 99; (ii) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 100; and (iii) ) A heavy chain variable region (VH) comprising CDR-H3 comprising the amino acid sequence of SEQ ID NO: 101; (b) (i) VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 96; ( Any one of claims 1 to 23, comprising: ii) a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 97; and (iii) a light chain variable region (VL) comprising CDR-L3 comprising the amino acid sequence of SEQ ID NO: 98. The method described in paragraph 1. The EGFRvIII binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 9 and a VL comprising the amino acid sequence of SEQ ID NO: 10, and the CD3 binding domain comprises a VH comprising the amino acid sequence of SEQ ID NO: 102, and a VH comprising the amino acid sequence of SEQ ID NO: 103. 25. The method according to any one of claims 1 to 24, comprising a VL comprising an amino acid sequence of 26. The method of any one of claims 1-25, wherein the EGFRvIII binding domain comprises the amino acid sequence of SEQ ID NO: 11 and the CD3 binding domain comprises the amino acid sequence of SEQ ID NO: 104. 27. The method of any one of claims 1-26, wherein the anti-EGFRvIII drug comprises the amino acid sequence of SEQ ID NO: 12. 28. The method of any one of claims 1-27, wherein the anti-EGFRvIII drug comprises the amino acid sequence of SEQ ID NO: 13. A kit comprising a delivery system and an anti-EGFRvIII agent, wherein one or more of the components of the delivery system, or portions thereof, that contact the aqueous solution are substantially free of polyvinyl chloride (PVC) or PVC plasticizer, and /or a kit substantially devoid of air. 30. The kit of claim 29, wherein the administration system is an infusion system including an infusion line, at least a portion of the infusion line being substantially devoid of PVC or PVC plasticizer. A kit comprising an IV bag containing an aqueous solution containing IVSS and physiological saline, and a container containing an anti-EGFRvIII drug, wherein at least a portion of the IV bag that contacts the aqueous solution is made of polyvinyl chloride (PVC) or PVC. Kit substantially devoid of plasticizers.

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